Non-coding RNA regulation of sex differences in stroke

中风仍然是全球第二大死亡原因，也是导致全球长期残疾的主要原因。 我们。尽管临床前研究已经确定了数百种潜在的药物制剂，但唯一的药理作用 可用的治疗方法仍然是早期溶栓治疗。翻译失败的一个主要因素可能是 在临床前研究中大量使用年轻雄性啮齿动物，缺乏针对性别的设计和分析 在临床试验中。中风是一种性两形性疾病，其发病率、患病率和性别差异 结果。线粒体在雄性和雌性脑缺血后的细胞死亡中都起到了中介作用，但方式不同。 非编码RNA，包括microRNAs(MiRs)，是调控细胞存活和 线粒体功能。在年轻的成年雄性动物中，MIR已被确定为 细胞对中风的反应，然而，它们在雌性或老年动物队列中的作用代表了一项关键知识 差距。我们之前的研究(2，4)和初步证据表明，女性和 男性卒中后表达选择的MIR及其靶点。这些目标的首要假设 MiR-181a和miR-200c表达和功能是卒中性二型性的关键决定因素 结果。为了测试这一点，我们将首先比较老年(20个月大)雌性和雄性小鼠的大脑中 建立实验性卒中大鼠大脑中动脉闭塞(MCAO)模型，比较原代雌性和雄性神经元及 利用体外缺血培养星形胶质细胞。在老年动物身上的初步证据显示，只有适度的后遗症 MCAO miR-181a反应仅限于女性，而相比之下，显著的miR-200C反应 在两性中都会发生。此外，我们的初步证据表明miR-181a和miR-200c存在性别差异。 基因打靶。在目标1中，我们将首先比较抗miR-181a或抗miR-181a或抗miR-181a的保护与MCAO IV后的治疗。 老年男性和女性的MIR-200C。然后，我们将评估长期的细胞特异性和性别特异性反应 在miR-181a和miR-200c中对MCAO。接下来，我们将比较MCAO后miR-200C对Long-MCAO的持续抑制。 老年男性和女性之间的神经康复术语，根据初步证据表明女性- MiR-200C特异性靶向神经营养蛋白reelin。在目标2中，我们将：1)重点关注miR- 181A和miR-200C在卒中后线粒体局部(核心和半影区)断裂方面的差异 2)确定miR-181a和miR-200c在细胞类型中的作用， 以及胞浆和线粒体中氧化磷酸化和破坏的性别特异性改变- 体外缺血后的处理。在目标3中，我们将首先定义细胞类型和性别相关的差异基因 以miR-181a(GRP78和XIAP)和miR-200C(XIAP，sirtuin-1，reelin)为靶点。最后，我们将确定是否 观察到的差异基因靶向可能归因于RNA甲基化的差异，以及这是否 对受伤的反应的变化。阐明了决定人类性二型性的分子机制 卒中对于克服翻译障碍和推进新的卒中治疗方法的发展是必要的。

Stroke remains the second leading cause of death worldwide and the primary cause of long-term disability in the US. Although pre-clinical studies have identified hundreds of potential drug agents, the only pharmacological treatment available remains early reperfusion with thrombolytics. A major factor for translational failure may be the overwhelming use of young male rodents in pre-clinical research, and lack of sex-specific design and analysis in clinical trials. Stroke is a sexually dimorphic disease with sex differences in incidence, prevalence, and outcome. Mitochondria mediate post-ischemia cell death in both males and females, but in different manners. Non-coding RNAs, including microRNAs (miRs), are upstream regulators of genes that regulate cell survival and mitochondrial function. In young adult male animals, miRs have been established as central regulators in the cellular response to stroke, however their role in females or aged animal cohorts represents a critical knowledge gap. Our prior studies (2, 4) and preliminary evidence suggest age-related differences between females and males in expression of select miRs and their targets following stroke. The overarching hypothesis of these Aims is that expression and function of miR-181a and miR-200c is a critical determinant of sexual dimorphism in stroke outcomes. To test this, we will first compare aged (20 month old) female and male mice using the middle cerebral artery occlusion (MCAO) model of experimental stroke, then compare primary female and male neuronal and astrocyte cultures utilizing in vitro ischemia. Preliminary evidence in aged animals reveals only a modest post- MCAO miR-181a response that is limited to females, while comparatively, a pronounced miR-200c response occurs in both sexes. Moreover, our preliminary evidence implies sex-differences in miR-181a and miR-200c gene targeting. In Aim 1, we will first compare protection with post-MCAO IV treatment of anti-miR-181a or anti- miR-200c in aged males and females. Then, we will assess the long-term cell-specific and sex-specific response in miR-181a and miR-200c to MCAO. Next, we will compare sustained post-MCAO miR-200c inhibition on long- term neuro-recovery between aged males and females, with the basis that preliminary evidence implies female- specific targeting of the neurotrophic protein reelin by miR-200c. In Aim 2, we will: 1) focus on the roles of miR- 181a and miR-200c in differences in regional (core versus penumbra) post-stroke disruption in mitochondrial function between aged males and females; and, 2) determine the roles of miR-181a and miR-200c in cell-type, and sex-specific alterations in oxidative phosphorylation and disruptions in cytosolic and mitochondrial Ca2+- handling following in vitro ischemia. In Aim 3 we will first define cell-type and sex-dependent differential gene targeting by miR-181a (Grp78 and XIAP) and miR-200c (XIAP, sirtuin-1, reelin). Finally we will determine whether observed differential gene targeting could be attributed to differences in RNA methylation, and whether this changes in response to injury. Delineating the molecular mechanisms that determine sexual dimorphism in stroke is necessary to overcome translational barriers and advance the development of novel stroke treatments.